It has been found quite useful to know the levels of phospholipids in biological fluids, generally. For example, phospholipids, particularly lecithin, are found in various biological membranes. Further, the phospholipid phosphatidylglycerol, as more fully described below, is found in amniotic fluid and can be used as an indicator of the lung maturity of the fetus. Other phospholipids besides lecithin (phosphatidylcholine) and phosphatidylglycerol which can be detected employing the method disclosed herein are, for example, phosphatidylinositol, phosphatidylserine and phosphatidylethanolamine. Although the present invention will be described in terms of its most preferred embodiment, that is, the determination of phosphatidylglycerol levels in amniotic fluid, the present invention can be used to determine the levels of any of the above-recited phospholipids in biological fluids, generally.
Proper functioning of the pulmonary system is essential for the fetus to survive in an extrauterine environment. Infants born with respiratory difficulties are said to have respiratory distress syndrome (RDS). The primary etiological defect in respiratory distress syndrome is a deficiency of surfactant, a complex mixture of lipids, proteins, and carbohydrates essential to the proper functioning of the mature lung. In the mature lung, phospholipids comprise 90-95% of the lipids. The major surface active phospholipid found in the surfactant is dipalmitoyllecithin. The second major surface active phospholipid is phosphatidylglycerol.
The most direct means of prenatally assessing fetal pulmonary maturity is measuring the production of lung surfactant phospholipids such as phosphatidylcholine (lecithin) and phosphatidylglycerol.
It has been determined that as pregnancy progresses, the sphingomyelin level in the surfactant remains relatively constant, while the lecithin level continues to increase, showing a very sharp increase after the 35th week of gestation. In the mature lung, lecithin comprises at least 50% of the total surfactant lipids. The constant level of sphingomyelin provides an internal reference for comparison with the surface active lecithin, thus providing the basis for the lecithin to sphingomyelin ratio (L/S) test developed by Gluck et al. as described in Am. J. Obstet. Gynecol., 109: 440 (1971).
Recent studies by Hallman et al. reported in Am. J. Gynecol., 125: 613 (1977), Tsai et al., Clin. Chem., 25: 682 (1979), Gotelli et al., Clin. Chem., 24: 1144 (1978), and Cunningham et al., Am. J. Obstet. Gynecol., 131: 719 (1978), indicate that measurement of phosphatidylglycerol may be of value in determining fetal pulmonary maturity. As alluded to previously, phosphatidylglycerol appears during the 35th-38th gestational week and has a good linear correlation with the L/S ratio. More to the point, Gluck, as reported in Clin. Chem., 23: 1107 (1977), points out that only after the appearance of phosphatidylglycerol in amniotic fluid is delivery safe in diabetic mothers. It was also discovered that the presence of blood or meconium in amniotic fluid affects the lecithin to sphingomyelin (L/S) ratio but not the level of phosphatidylglycerol. Although the L/S ratio test has gained wide acceptance as the most reliable prognostic index of fetal pulmonary maturity in most pregnancies, the results must be interpreted with caution for certain maternal complications such as diabetes mellitus, hypertension, severe anemia and intrinsic renal disease, can adversely affect the L/S ratio readings.
It was therefore found desirable to find alternative methods of assessing fetal pulmonary maturity, which are relatively fast, specific and require a minimum amount of skill, experience and sophisticated instrumentation to gain results with high precision and accuracy. It was the development of these alternative methods which led to the present invention for the determination of the presence of phospholipids in a biological fluid. The prior art has used biochemical quantitation and biophysical measurements as techniques for evaluating amniotic fluid surfactant. All of the prior art methods, however, suffer from either being overly time consuming and tedious, and requiring skill and expertise to obtain reasonably high precision, and requiring the use of hazardous chemicals and highly sophisticated and expensive instrumentation or in providing methods which are simply non-specific. The present invention provides a method exhibiting none of these drawbacks.